Questions tagged [rna-seq]

Questions should include this tag if they pertain to issues related to bioinformatics analysis of RNA-seq data, e.g. normalization, differential expression analysis, sequencing and experimental design or transcriptome assembly...

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16 views

Annatation with cuffdiff in RNA_Seq Analysis

my annotation file is in .gff format I would like to convert it to .gtf format or if there is a way to directly download the annotation file in .gtf format? I am working on sequences from the P ...
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3answers
1k views

RIP-seq analysis?

Given an experiment consisting of an input (baseline RNA) and IP (pulldown to find RNAs bound to certain protein of interest)... Is a DE analysis performed over the RNA-seq data from the samples (lets ...
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73 views

Error in my RNAseq analysis

I was following the RNAseq analysis tutorial online(https://combine-australia.github.io/RNAseq-R/06-rnaseq-day1.html) but am not obtaining the same variance values for each row in the logcounts matrix....
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10 views

include a glimma interface in a shiny app

I am trying to code a shiny app for RNA-Seq data analysis. I would like to include glimma interactive plots in it. However, in my current interface, clicking the action button ...
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0answers
38 views

Normalization for microbiome 16s sequence analysis

The way I understand things, normalization (such as in DeSeq2, EdgeR, etc.) serves two purposes: 1) Model the "real" abundance in the original samples from the read counts, 2) Make the abundance ...
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1answer
16 views

mapping and de novo assembly of plant without reference genome

I'm studying on a plant that has no reference genome and only has one scaffold assembly and one gff3 annotation file. Can I create an index with the same assembly and gff3 in STAR and do the mapping? ...
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3answers
112 views

Which correlation method to compute the correlation score between different clusters of Sc-RNAseq data?

I'm analyzing single cell rna-seq data and trying to compute the correlation score between different clusters. Wondering how to choose the correlation method("pearson" (default), "kendall", or "...
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0answers
14 views

What should I do when I have “#NAME?” in my Log Fold Change data to calculate Z-score? replace with zero or what?

I am trying to calculate the Z-score for several columns with Log fold change (LFC) data from RNA-seq expression values. But the mean for all columns, except one of them, is -inf and the standard ...
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1answer
43 views

RNASeq: Normalization, stabilization, gene length and rlog

I was thinking about the best method for normalization, which takes gene length into account (in order to compare genes)... Do you think I can do that? : - taking raw counts and dividing each gene by ...
0
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1answer
44 views

how to interpret of “pvclust” dendrogram and finding height for cutting dendrogram?

I study on RNA-seq expression dataset about one cancer in TCGA. I downloaded FPKM dataset and removed batch effect by ComBat() function. Now, I used pvclust for ...
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18 views

How to detail the specific GO terms

How can I get more specific GO terms when using clusterProfiler? I got my Dotplot by: ...
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24 views

Model matrix design for limma-voom and batch effect correction

I've posted it on Bioconductor but didn't get a response, so I thought maybe I could get some help here (most likely the same audience but I'll try) I have multiple clusters in a 270 leukaemia sample ...
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1answer
26 views

Pre-filtering genes for Principal Component Analysis

I have a raw counts data-set of 20,502 genes and 137 samples. I want to find out Principal Components which best explain variation between samples in different stages of tumor. I am new to Machine ...
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2answers
34 views

about GO terms's name

Did anyone know whether the GO terms can include more detail information? Like I can get the DotPlot of the GO terms as below: The problem is that some of the genes, in the GO terms, is more about ...
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1answer
48 views

Where can I get ensembl Medaka genome for RNAseq

I am trying to map a RNA-seq dataset using ensembl genome for medaka fish. From here http://uswest.ensembl.org/Oryzias_latipes/Info/Index when I click on a ...
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1answer
47 views

CIBERSORT runtime error eval failed

I was running CIBERSORT but caught this error right after it started permutation: ...
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2answers
407 views

Reads mapped to exonic, intronic and intergenic regions

After the alignment step I checked the rnaseq metrics of all the samples. Among 40 samples three samples show high percentage of reads mapped to intronic regions. What could be the reason? ...
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0answers
28 views

Should I use log2-CPM values (voom-limma) as input for my model?

We have created a model to integrate several OMICs data, but we realized that the maximum TPM values of RNA-Seq data were so big that had unexpected effects on our results. We hypothesized that this ...
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0answers
75 views

Error using bseqsc

I will be very grateful for any hint on how to overcome the error. I wish to deconvolve my bulk RNA seq data obtained from the lungs of mice using single cell RNA seq data. For practice, I am ...
7
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2answers
656 views

Convert R RNA-seq data object to a Python object

I have done some work in R and would like to try a Python tool. What is a good way to import the data (and its annotations etc) as a Python object? I am particularly interested in converting a ...
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0answers
25 views

How to analyze expression of certain group of genes in certain types of cell?

I need to analyze expression of ~ 400 genes with a certain function in the embryonic stem cells. All these genes are combined into one database with RefSeq IDs. What is a recommended workflow to ...
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1answer
62 views

Is re-normalization of RNAseq data recommended for analysis of gene subsets?

I downloaded an RNAseq dataset from TCGA database in 3 formats: 1) HTSeq counts; 2) FPKM; 3) FPQM-upper quartile normalized. The complete dataset contains ~60,000 genes. All of my analysis will focus ...
1
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1answer
57 views

RNAseq data analysis

I am currently doing a Differential Gene Expression of RNAseq data in Lung Adenocarcinoma using TCGAbiolinks. In the data Preprocessing step TCGAanalyze_Normalisation, I am confused as to which method ...
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1answer
47 views

How I deal with this expression set?

I have a gene expression raw counts like below for 16 patients ...
2
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1answer
84 views

How I know which gene is a good predictor in this neural network or not?

I have 25 highly differentially expressed genes among and patients to chemotherapy. I have made a neural network of these genes. Accuracy of model is 0.73 but I don't know from 25 genes how I could ...
2
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2answers
157 views

TPM or rlog(CPM) for comparing expression?

I want to see the expression of a gene in a group of patient amongst the entire cohort using my RNA-Seq data. While I can do a differential expression analysis with limma or DESeq2, I want to see how ...
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1answer
31 views
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30 views

How to segment genome into homozygote/heterozygote blocks based on RNA-Seq data

Given a RNA-Seq data of a progeny between two inbred lines (for example an F4 plant which was derived from two inbred lines (parents) and the F1 selfed 3 times; however this is probably not crucial ...
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2answers
112 views

too long header for fasta file

I have a fasta file, like this: ...
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1answer
79 views

How to transform and sort the matrix to make a heatmap showing signatures?

I have a matrix data with cells as rows and samples as columns. Here I am giving the data with dput ...
2
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1answer
87 views

sva for RNA-Seq data without known phenotype

I have been working on RNA-Seq data from two different cohorts, and they show very strong batch effect (~35% variance explained by 1st component in PCA). Since I am trying to do a class discovery from ...
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48 views

Selecting genes with more contribution from PCA

I have RNA-seq data in response to treatment vs non response; By machine learning I selected three principle components likely can predict the response based on the gene expression. Now I have ...
2
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3answers
86 views

Calculating p-value for introns which are retained (or not) between 2 conditions

I am trying to calculate p-values and ultimately FDR values for RI introns for 2 conditions of experiment I have(control and knockdown) For every condition I have 3 biological replicates each: 3 ...
4
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2answers
674 views

Seurat for clustering bulk RNA-seq?

Is it ever ok to use Seurat for clustering bulk samples? I am looking at FPKM data from ~750 bulk RNA-seq samples generated using Cufflinks. As suggested for FPKM ...
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1answer
53 views

Convert FPKM to RPKM

I have my 24 samples analyzed by Hisat2>Stringtie, from paired end sequenced data. I have got FPKM data and am planing to convert them to RPKM. to be able to compare my samples with another already ...
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1answer
83 views

How I deal with this kind of gene expression comparision

I have RNA-seq from two sequencing batches; Lab technician says that he has run the RNA expression quantification two times in bathes 1 and 2 for example tumor 1 in batch 1 and tumor 1 in batch 2 , ...
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1answer
17 views

How to map selected genes to Metabolic pathway Maps

I have a selected Arabidopsis Genome Initiative (AGI) list for RNA seq and proteomics data, how can I map them to metabolic pathway maps to vilualize(e.g. TCA cycle / FA / Photosynthesis) in KEGG or ...
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1answer
163 views

Changing the axis limits of ggplot objects

By EnhancedVolcano R package I have this plot with this code ...
2
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2answers
50 views

Find most similar genes to a set of genes of interest in time series RNA-seq data

I have gene expression data (RNA-seq) for 30 different time points (from 0 to 60 min each 2 min). I have a set of 8 genes that behave similarly (although not identically) and I want to find the top X ...
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2answers
95 views

Interpreting this PCA plot for RNA-seq

I have RNA-seq from two sequencing batches; Lab technician says that he has run the RNA expression quantification two times in bathes 1 and 2 for example ...
0
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2answers
28 views

In-sample and across samples normalized expression

I want to get the expression data that is in-sample normalized like FPKM and also across samples normalized as obtained using DESeq2 or else. What I am currently doing is that I first normalize the ...
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0answers
53 views

Module preservation analysis WGCNA

I have done a WGCNA (weighted gene co-expression network analysis) analysis for various brain disorders. So along with control, I have 3 different disorders such as BPD, MDD and SCZ. Now I have to ...
2
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2answers
60 views

How to best detect the “peaks” in RNA-seq data that are not assigned to any gene?

I encountered that many reads from single-cell RNA seq data were lost in the analysis because not assigned to any gene (genome: galgal6). I am trying to find an approach than could give me all the "...
2
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1answer
126 views

How to input data for DESeq2 from individual HTSeq count?

I am comparing the gene expression of 2 bacteria under 1 condition. I have now the count tables for 3 tech. replicates for each bacteria. ...
17
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2answers
302 views

Confirm success or failure of RNA-Seq normalization

I am working with a set of (bulk) RNA-Seq data collected across multiple runs, run at different times of the year. I have normalized my data using library size / quantile / RUV normalization, and ...
5
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3answers
121 views

Show presence of known mutation in RNA-seq data

We have RNA-seq fastq data from control (WT) patients and a patient with a point mutation at a known location in one gene. I'd like to retrieve the reads aligning to that gene and show the presence of ...
0
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1answer
35 views

RNA-seq: How to get new expression count after normalization

I've RNA seq, Human, Paired-end data, Sample size is <40. These are aligned using STAR, RSEM processed. With RSEM I've TPM and expected counts, that is two files columns as individual IDs and row ...
8
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1answer
2k views

How to apply upperquartile normalization on RSEM expected counts?

I see that TCGA RNASeq V2 RSEM data is normalized with upper-quartile normalization. After doing Quantification with RSEM with the samples I have, I got "genes.results" as output which has gene id, ...
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0answers
51 views

length of 'dimnames' [1686] must match that of 'dims' [3]

Please if anyone has experience with the use of the BSEQ-SC package for the deconvolution of bulk RNA sequencing data with single cell RNA sequencing data I will be very grateful for your suggestion. ...